Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation.

The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.

A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos.

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency.

Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.

Deep Learning Neural Networks Highly Predict Very Early Onset of Pluripotent Stem Cell Differentiation.

Deep learning is a significant step forward for developing autonomous tasks. One of its branches, computer vision, allows image recognition with high accuracy thanks to the use of convolutional neural networks (CNNs). Our goal was to train a CNN with transmitted light microscopy images to distinguish pluripotent stem cells from early differentiating cells. We induced differentiation of mouse embryonic stem cells to epiblast-like cells and took images at several time points from the initial stimulus. We found that the networks can be trained to recognize undifferentiated cells from differentiating cells with an accuracy higher than 99%. Successful prediction started just 20 min after the onset of differentiation. Furthermore, CNNs displayed great performance in several similar pluripotent stem cell (PSC) settings, including mesoderm differentiation in human induced PSCs. Accurate cellular morphology recognition in a simple microscopic set up may have a significant impact on how cell assays are performed in the near future.

Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system.

OBJECTIVES: The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS: Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.

Diferenciación en cardiomiocitos a partir de células madre embrionarias humanas / Differentiation of Human Embryonic Stem Cells into Cardiomyocytes

Introducción Las células madre son motivo de intensa investigación debido a la posibilidad de su utilización en el tratamiento de numerosas enfermedades, en particular las cardiovasculares. La diferenciación de células madre embrionarias humanas en cardiomiocitos se ha realizado exitosamente in vitro. Se han establecido métodos de cultivo y diferenciación, señales involucradas en la cardiogénesis y los cardiomiocitos generados se han utilizado en modelos de regeneración miocárdica. Sin embargo, aún quedan muchos interrogantes que se están investigando activamente. Objetivo Desarrollar una metodología que permita el cultivo de células embrionarias y su diferenciación en cardiomiocitos. Material y métodos Se utilizaron cuatro líneas de células madre embrionarias humanas. Se cultivaron y diferenciaron a través de los métodos publicados previamente en la bibliografía. El estado indiferenciado y la diferenciación en cardiomiocitos se verificaron por medio de inmunomarcación fluorescente y RT-PCR. Resultados La metodología utilizada permitió cultivar las células y mantenerlas en estado indiferenciado. Aunque con eficacia dispar, se logró la diferenciación en cardiomiocitos de las cuatro líneas celulares utilizadas. La confirmación se realizó por medio de la expresión de factores de transcripción miocárdicos y proteínas estructurales cardíacas. Conclusiones El cultivo y la diferenciación de células madre embrionarias humanas fue posible en nuestro sistema. Estos resultados preliminares nos impulsan a continuar y a desarrollar nuestros métodos con células pluripotentes inducidas.

Background The role of stem cells in the treatment of several conditions, especially heart diseases, is under permanent investigation. Human embryonic stem cells have been successfully differentiated in vitro into cardiomyocytes. Methods of cell culture and cardiomyocyte differentiation are well established; signals regulating cardiogenesis have been identified and the cardiomyocytes generated have been used in models of myocardial regeneration. However, several questions still remain and are currently under active investigation. Objective To develop a culture system that is suitable for the induction of embryonic stem cells to cardiomyocyte differentiation. Material and Methods Four human embryonic stem cell lines were used. The cells were cultured and differentiation was induced using methods previously described. The presence of cells in an undifferentiated state and cardiomyocyte differentiation was detected by immunohistochemical studies (fluorescent staining) and RT-PCR. Results The methodology used allowed stem cells growth in the culture, and maintained them in an undifferentiated state. Cardiomyocyte differentiation was achieved in the four cell lines used, yet with uneven efficacy. This was confirmed by the expression of myocardial transcription factors and heart structural proteins. Conclusions Our system allowed human embryonic stem cell growth and differentiation in the culture. These preliminary results encourage us to continue developing our methods with induced pluripotent stem cells.

Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways.

The potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of alpha isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.

Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression.

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator.

Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5'-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5'-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.